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Molecular cloning, expression, and primary sequence of outer membrane protein P2 of Haemophilus influenzae type b
1Edward Mallinckrodt Department of Pediatrics, Washington University, School of Medicine, St. Louis, Missouri.
Abstract:
The structural gene for the porin of Haemophilus influenzae type b, designated outer membrane protein P2, was cloned, and the DNA sequence was determined. An oligonucleotide probe generated by reverse translation of N-terminal amino acid sequence data from the purified protein was used to screen genomic DNA. The probe detected a single EcoRI fragment of approximately 1,700 base pairs which was cloned to lambda gt11 and then into M13 and partially sequenced. The derived amino acid sequence indicated that we had cloned the N-terminal portion of the P2 gene. An overlapping approximately 1,600-base-pair PvuII genomic fragment was cloned into M13, and the sequence of the remainder of the P2 gene was determined. The gene for P2 was then reconstructed under the control of the T7 promoter and expressed in Escherichia coli. The N-terminal sequence of the purified protein corresponds to residues 21 through 34 of the derived amino acid sequence. Thus, the protein is synthesized with a 20-amino-acid leader peptide. The Mr of the processed protein is 37,782, in good agreement with the estimate of 37,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Insights
The porin (outer membrane protein P2) gene from Haemophilus influenzae type b was cloned and sequenced. This revealed a 20-amino-acid leader peptide, crucial for understanding bacterial outer membrane protein synthesis.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Haemophilus influenzae type b is a significant human pathogen.
- Outer membrane proteins, such as porin P2, play critical roles in bacterial structure and function.
- Understanding the genetic basis of these proteins is essential for developing targeted therapies.
Purpose of the Study:
- To clone and determine the DNA sequence of the structural gene for Haemophilus influenzae type b porin (outer membrane protein P2).
- To characterize the P2 protein, including its N-terminal sequence and processing.
- To enable the expression of the P2 gene in a heterologous system.
Main Methods:
- Oligonucleotide probing based on N-terminal amino acid sequence of purified P2 protein.
- Genomic DNA screening using EcoRI and PvuII restriction fragments.
- Cloning into lambda gt11 and M13 vectors for DNA sequencing.
- Gene reconstruction under T7 promoter for expression in Escherichia coli.
Main Results:
- The complete DNA sequence of the P2 gene was determined.
- The derived amino acid sequence revealed a 20-amino-acid leader peptide.
- The mature P2 protein has an Mr of 37,782, consistent with SDS-PAGE estimates.
- The N-terminal sequence of the purified protein matched residues 21-34 of the deduced sequence.
Conclusions:
- The structural gene for Haemophilus influenzae type b outer membrane protein P2 has been successfully cloned and sequenced.
- The P2 protein is synthesized as a precursor with a 20-amino-acid leader peptide.
- This work provides a foundation for further studies on porin structure-function and potential therapeutic targets.